Systemic Metabolic Rewiring in a Mouse Model of Left Ventricular Hypertrophy

Figures

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  Figure 1. (A) IDC data from WT (N = 5) and Mybpc3−/− (N = 7) males are represented as a moving average of RER. The average RER of Mybpc3−/− males (AUC = 8.473 ± 0.08646) was higher than WT males (AUC = 6.553 ± 0.09763) over the course of 48 h. Error bars (shaded region) represent SD. (B) Individual RER values from each male mouse cohort were restricted to one light cycle (16–28 h) for cycle-specific statistical analysis. Mybpc3−/− males (mean = 0.8773) had significantly higher RER readings compared to WT males (mean = 0.8079) during the light cycle (unpaired t-test; p < 0.0001). Error bars represent SEM. (C) Individual RER values from each male mouse cohort were restricted to one dark cycle (6–16 h) for cycle-specific statistical analysis. Mybpc3−/− males (mean = 0.9398) had significantly higher RER values during the dark cycle compared to WT males (mean = 0.9305; unpaired t-test; p = 0.0137). Error bars represent SEM. (D) Female IDC data from WT (N = 5) and Mybpc3−/− (N = 5) mice are represented as a moving average of RER. The average RER of Mybpc3−/− females (AUC = 7.911 ± 0.1097) was similar to WT females (AUC = 7.563 ± 0.09611) over the course of 48 h. Error bars represent SD. (E)…

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  Figure 2. (A) Male Mybpc3−/− mice (N = 17) had significantly lower BW mass compared to age-matched WT males (N = 12; unpaired t-test; p < 0.0001). (B) Male Mybpc3−/− mice (N = 17) had significantly lower EFP mass compared to age-matched male WT mice (N = 12; unpaired t-test; p < 0.0001). (C) Male EFP weights were normalized to BW to calculate an EFP/BW ratio to serve as an estimation of body composition. An unpaired t-test showed that male Mybpc3−/− mice had a significantly lower EFP/BW ratio compared to WT males (unpaired t-test; p < 0.0001). (D) There was no difference in female Mybpc3−/− mice (N = 12) BW mass compared to age-matched WT females (N = 6; unpaired t-test; p = 0.5803). (E) There was no significant difference between female Mybpc−/− mice (N = 12) POAT mass compared to age-matched female WT mice (N = 6) POAT mass (unpaired t-test; p = 0.4091). (F) Female POAT weights were normalized to BW to calculate a POAT/BW ratio to serve as an estimation of body composition. An unpaired t-test showed that female Mybpc3−/− and female WT mice had no significant difference in the POAT/BW ratio (unpaired t-test; p = 0.4091). **** = p < 0.0001. All error bars represent SD.

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  Figure 3. Histological analysis was performed in cardiac tissue collected from male Mybpc3−/− and WT mice. (A) H&E staining shows that Mybpc3−/− mice have differences in cardiomyocyte size and organization. (B) MT staining shows that Mybpc3−/− mice have clear, large regions of collagen deposition. (C) ORO staining shows that Mybpc3−/− cardiac tissue has an accumulation of large lipid droplets (as indicated by arrows). (D) PAS staining indicated glycogen-positive cardiomyocytes in both WT and Mybpc3−/− cardiac tissues. Scale bar represents 100 μM; N = 3 for each group. (E) The number of lipid droplets per 600 μM in Mybpc3−/− mouse (N = 15) cardiac tissue was significantly higher compared to WT mice (N = 15; unpaired t-test; p < 0.0001). (F) The number of glycogen granules per 600 μM was significantly higher in Mybpc3−/− (N = 15) cardiac tissue compared to WT mice (N = 15; unpaired t-test; p = 0.0460). * = p < 0.05; **** = p < 0.0001. All error bars represent SD.

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  Figure 4. Histological analysis was performed in liver tissue collected from male Mybpc3−/− and WT mice. (A) H&E staining shows no clear differences in hepatocyte size or organization. (B) MT staining shows that Mybpc3−/− mice have clear, large regions of collagen deposition. (C) ORO staining shows that Mybpc3−/− hepatic tissue has an accumulation of large lipid droplets. (D) PAS staining indicated glycogen-positive cardiomyocytes in both WT and Mybpc3−/− cardiac tissues. Scale bar represents 100 μM; N = 3 for each group. (E) Mybpc3−/− mice (N = 15) had significantly more lipid droplets in the liver per 600 μM compared to WT mice (N = 15; p < 0.0001). **** = p < 0.0001. Error bars represent SD.

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  Figure 5. (A) Two-way ANOVA mixed-effects analysis was performed to determine the effect of fasting on blood glucose levels in Mybpc3−/− (N = 8) vs. WT (N = 8) male mice. Overall, fasting only decreased blood glucose levels in WT mice. Fed WT mice had significantly higher blood glucose levels compared to Mybpc3−/− mice (2-way ANOVA; p < 0.0001). (B) Male Mybpc3−/− had significantly higher blood ketone levels compared to WT mice in both a fed (2-way ANOVA; p < 0.00001) and fasted (2-way ANOVA; p < 0.00001) state. WT mice had a significant increase in blood ketone levels in a fasted state compared to a fed state (2-way ANOVA; p < 0.0001). (C) Fold change (FC) was calculated for each acyl-CoA species by dividing the mean Mybpc3−/− concentration by the mean WT concentration. Values over 1.0 indicate an increase in Mybpc3−/− mice; values below 1.0 indicate a decrease in Mybpc3−/− mice. p-values were calculated using the mean of each genotype to determine significant increases/decreases. (D–H) All acylcarnitine chain lengths with significant differences in mean concentrations are shown. * = p < 0.05; ** = p < 0.005; *** = p < 0.0005; **** = p < 0.0001. Error bars represent SD.

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  Figure 6. (A) RER values within each genotype were averaged and plotted as a moving RER average against time. Unpaired t-tests to compare WT and Mybpc3−/− mean at each time point (X-axis) were performed. (B) Blood glucose levels measured pre- and post-exhaustion were plotted for Mybpc3−/− male mice (N = 5) and WT mice (N = 5). A paired t-test showed that WT mice had a significant decrease in blood glucose levels post-exhaustion (p = 0.0009). A paired t-test showed that Mybpc3−/− mice (N = 5) had a significant increase in blood glucose levels post-exhaustion (p = 0.0429). An unpaired t-test showed that Mybpc3−/− mice had significantly lower blood glucose levels than WT mice pre-exhaustion (p = 0.0002). (C) Blood ketone levels measured pre- and post-exhaustion were plotted for Mybpc3−/− male mice (N = 5) and WT mice (N = 5). Mybpc3−/− mice had significantly higher blood ketone levels pre-exhaustion compared to WT mice (p < 0.0001). Mybpc3−/− mice had significantly lower blood ketone levels compared to WT mice post-exhaustion (p < 0.0001). WT mice had significantly higher blood ketone levels post-exercise challenge (p = 0.0327). (D) Mybpc3−/− mice ran a significantly shorter distance compared…

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  Figure 7. (A) The mean respiration of WT mtLV (N = 6) and Mybpc3−/− mtLV (N = 6) is represented as a moving average in response to acute treatment of various metabolic substrates. AUC analysis showed that WT mtLV had an AUC value of 111.20 ± 8.23 compared to the mtLV Mybpc3−/− AUC value of 42.48 ± 3.08. Unpaired t-test analysis showed that Mybpc3−/− mtLVs had significantly lower mitochondrial respiration in response to pyruvate (Q = 0.000111), glutamate (Q < 0.000001), succinate (Q = 0.000090), CCCP (p = 0.000090), and rotenone (Q = 0.000632) treatment. (B) Complex-specific activity was calculated in WT (N = 6) and Mybpc3−/− (N = 6) mtLV. Mybpc3−/− mtLV had significantly reduced CI-linked (p < 0.0001), CI + CII-linked (p < 0.0001), max OXPHOS (p < 0.0001), and CII-linked (p < 0.0001) compared to WT mtLV. (C) Complex-specific activity was calculated from mean oxygen consumption in mtQuad isolated from WT mice (N = 7) and Mybpc3−/− mice (N = 7) quadriceps. Unpaired t-tests showed that mtQuad from Mybpc3−/− mice was significantly increased in CI + CII-linked (p = 0.0111), max OXPHOS (p = 0.0469), and CII-linked (p = 0.0030) compared to WT mtQuad. (D) Complex-specific activity was calculated…

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  Figure 8. (A) Metabolic flux is represented as oxygen flux in response to acute treatment of various substrates in permeabilized quadriceps muscle fibers (pQuad) collected from Mybpc3−/− mice (N = 4) and WT mice (N = 5). AUC analysis indicated that WT and Mybpc3−/− pQuad had similar area values, with 119.5 ± 5.664 and 132.5 ± 7.173, respectively. Mybpc3−/− pQuad had a significantly higher response to CCCP (Q < 0.0001) compared to WT mtQuad. (B) State III, maximum OXPHOS capacity, and complex-specific activity were calculated for pQuad collected from Mybpc3−/− and WT mice with treatment of a long-chain fatty acid (LCFA). Mybpc3−/− pQuad (N = 4) had significantly higher oxygen uptake only at maximal OXPHOS compared to WT pQuad (N = 5; p = 0.0089). (C) Metabolic flux is represented as oxygen flux in response to acute treatment of various substrates, including an LCFA, in permeabilized soleus muscle fibers (pSoleus). AUC analysis showed that the total peak area in Mybpc3−/− pSoleus was higher than WT pSoleus, with 109.1 ± 4.557 and 65.7 ± 5.007, respectively. Mybpc3−/− pSoleus samples had significantly higher oxygen influx after fatty acid (Q = 0.0061), malate (Q = 0.0061), glutamate (Q =…

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  Figure 9. Mybpc3−/− mice and WT mice of both sexes were fed an HFD for 10 weeks. (A) Male Mybpc3−/− mice (N = 5) had a significantly lower body weight compared to WT male mice (N = 5; p < 0.000001). Female Mybpc3−/− mice (N = 4) had a significantly lower body weight compared to female WT mice (N = 4; p = 0.000131). (B) Male Mybpc3−/− mice (N = 5) had a significantly lower mean GFP (EFP) weight compared to WT male mice (N = 5; p = 0.000085). Female Mybpc3−/− mice (N = 4) had a significantly lower mean GFP (POAT) weight compared to WT mice (N = 4). (C) Male EFP weights were normalized to BW to calculate an EFP/BW ratio to serve as an estimation of body composition. Two-way ANOVA analysis showed that both male (N = 8) and female (N = 8) Mybpc3−/− mice had a significantly lower GFP/BW ratio compared to WT males (N = 8) and females (N = 7; F (1, 15) = 37.52; p < 0.00001). (D) A qualitative histology study was performed with H&E, MT, ORO, and PAS in heart tissue isolated from WT and Mybpc3−/− male mice fed an HFD for 10 weeks. (E) A qualitative histology study was performed with H&E, MT, ORO, and PAS in heart and liver tissue isolated from WT and Mybpc3−/− male mice fed an HFD for 10 weeks. ***…

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  Figure 10. WT and Mybpc3−/− mice were fed an HFD for 10 weeks. (A) Exercise-naïve Mybpc3−/− and WT mice were subject to an acute exercise challenge, reported as an RER moving average. AUC analysis was performed; WT mice (N = 7) had a peak area value of 6.835 ± 0.036, and Mybpc3−/− mice (N = 5) had a peak area value of 8.044 ± 0.023. Mybpc3−/− mice had a significantly higher RER at the beginning of the exercise challenge (Q = 0.0011; Q = 0.0011; Q = 0.0002; Q < 0.00001). (B) Two-way ANOVA analysis indicated that Mybpc3−/− mice (N = 5) had significantly lower blood glucose levels compared to WT mice (N = 7), both pre- and post-exercise challenge (F (1, 6) = 39.59; p = 0.0008). (C) Two-way ANOVA analysis indicated that Mybpc3−/− mice (N = 5) had significantly lower blood ketone levels compared to WT mice (N = 7), both pre- and post-exercise challenge. (D) There was no significant difference in mean distance run between Mybpc3−/− vs. WT mice (unpaired t-test; p = 0.0614). (E) Complex-specific activity was measured in mtLV collected from Mybpc3−/− and WT mouse hearts, represented as an oxygen flux. Mybpc3−/− mice (N = 5) had significantly lower CI-linked activity compared to control counterparts…

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  Figure 11. Schematic of the proposed mechanism that supports systemic metabolic rewiring in LVH mice. Loss of FAO OXPHOS in cardiac tissue leads to reduced LCFA uptake and utilization. The compromised cardiomyocytes increase glucose uptake to support energy production via glycolysis and glucose-linked OXPHOS. The liver takes up excess LCFAs for energy production, converting unused acetyl-CoA into ketone bodies to be used by other tissues. Slow-twitch skeletal muscles, which are metabolically reliant on OXPHOS, must metabolically shift to rely mostly on glucose catabolism to drive OXPHOS. Fast-twitch skeletal muscles shift to metabolically rely on ketone bodies generated by the liver. Down arrows (purple) indicate a decreased parameter in LVH contexts. Up arrows (orange) indicate an increased parameter in LVH contexts.